The key step in regulating the proliferation of cells and viruses is control of initiation of DNA replication. Central to understanding how this control works is understanding the nature of sites where replication begins (i.e. origins of DNA replication). We and others have developed various strategies for mapping the locations of initiation sites in the chromosomes of cells from metazoan animals to a resolution of 1 kilobase (kb). At least 18 specific initiation sites have now been mapped in mammalian chromosomes. In general, most initiation events at these sites occur within a 0.5 to 2 kb "origin of bidirectional replication" (OBR) although initiation events can be detected throughout a larger "initiation zone" of up to 60 kb. We have shown that site specific initiation of DNA replication results from the interaction between specific DNA sequences and nuclear (chromatin?) structure. Moreover, others have shown that specific initiation sites are both developmentally acquired and established during the mid to late G1-phase of their cell division cycle. We have developed an assay for cis-acting sequences required for site specific initiation by transfecting hamster cells with plasmids containing a well characterized OBR (ori-beta). These ectopic ori-beta sequences behave like active replication origins, while ectopic copies of sequences from the replication initiation inactive regions remain inactive. Therefore, origin activity depends on specific DNA sequences. Analysis of DNA methylation at two replication origins (ori-beta, ori-RPS14) revealed that both OBRs are associated with a high density cluster of methylated CpG dinucleotides throughout their cell division cycle. However, neither OBR contained the putative densely methylated island (DMI) reported by Tasheva and Roufa [Mol. Cell. Biol. 14: 5636-44, 1994] in which all cytosines are methylated in proliferating cells. Remethylation studies demonstrated directly that active OBR sequences contained the same methylation pattern as bulk OBR sequences. Other mammalian OBRs have the potential for similar methylation patterns, suggesting that these mCpG clusters may play a role in either the establishment or regulation of mammalian replication origins. We are now attempting to identify specific sequences that contribute to site specific initiation, and the proteins that interact with these sequences.